p53 is a tumor suppressor protein that is heavily involved in important cellular processes, most notably in cell cycle regulation, apoptosis and DNA repair. The full-length wild-type p53 protein binds to several DNA sequences, functioning as a sequence-specific transcriptional activator. Its flexible nature lends it the ability to interact with other proteins, exerting its function as a master regulatory protein and transcription factor. Its roles include maintaining genome integrity and regulating the cell cycle. As such, wild-type p53 is activated in response to cellular stress, such as uncontrolled cell growth, DNA damage and hypoxia. Its function is lost in more than 50% of human cancers with several mutations, all of which are often associated with the most pernicious manifestations of the disease (1). Thus, in recent years, p53 has taken on a pivotal role in the realm of cancer research and has been rendered a key target in the development of modern cancer therapeutics.
Human p53 is a homotetramer—a protein complex that is comprised of four identical monomers, each of which consist an N-terminal transactivation domain, a proline-rich domain, a tetramerization domain and a C-terminal regulatory domain (FIG. 1). Notably, it has been shown that the DNA-binding domain (DBD) of p53 is conformationally unstable; some 95% of p53 mutations occur within this region, which further decreases its stability and prompts protein unfolding or conformational transition (2). These mutations cluster into discernible ‘hot-spots’, which are sites of identifiable mutations within the DNA-binding domain. Of these many hot-spots disease mutants, two classes have been shown to have great clinical significance. The first class consists of structural mutations, which lead to the partial unfolding of the protein, rendering it functionally inactive. Structural mutants, such as R248Q, are present in approximately 30% of reported clinical cases of p53 mutations. Contact mutants, such as R248W, make up the second class and do not affect the conformational stability of the DNA binding domain. Instead, these mutants are unable to bind to DNA due to a missense mutation in an amino acid residue crucial for DNA interaction. Contact mutants account for approximately 20% of clinical cases3. Despite their mechanistic differences, both classes of these hot-spot disease mutants engender similar phenotypic consequences: loss-of-function, dominant-negative activity and gain-of-oncogenic function. Loss-of-function describes the destruction of p53 DNA-binding ability, which prevents the transcriptional activation of genes that initiate apoptosis or DNA repair. Dominant-negative activity transpires from the assimilation of wild-type p53 protein with its mutant form into tetrameric inclusions, thereby dominantly and negatively suppressing the wild-type protein function, effectively hindering its inherent tumor suppressor functionality. Gain-of-oncogenic function results from the incorporation of p53 paralogs p63 and p73 into these cellular inclusions, which further inhibits the transcription of target genes downstream of p53-activated transcription, ultimately leading to the promotion of tumor metastasis (2).
Previous research has shown that mutant p53 undergoes aggregation in vitro. Using TANGO, a predictive algorithm, residues 251-257 were found to be aggregation-prone. To determine whether these residues are critical for nucleating aggregation, residue 254 was mutated from isoleucine, a hydrophobic residue, to arginine, which is positively charged at physiological pH. Interestingly, not only was the aggregation propensity of the resulting mutant (I254R) nullified, but it was also found that in the presence of I254R, contact and structural mutants failed to aggregate (5).
More recently, it has been found that several p53 DBD mutants form amyloid-like aggregates in tumor cell lines and breast cancer biopsies (4). Being an early event in carcinogenesis, p53 inactivation through mutation is associated with poor response to treatment and high mortality rates. Missense mutations at the R248 site are among the most common in known p53-mutant cancers, including pancreatic carcinoma. Pancreatic cancers do not currently have standard targeted treatments and generally have extremely poor prognoses: it is the seventh most common cause of death from cancer worldwide, with a ten-year survival rate of just 1% (6).